Molecular mimicry between Anoctamin 2 and Epstein- Barr virus nuclear antigen 1 associates with multiple sclerosis risk Katarina Tengvalla,b,1, Jesse Huanga,b, Cecilia Hellströmc, Patrick Kammerd, Martin Biströme, Burcu Ayogluf, Izaura Lima Bomfima,b,PernillaStridha,b, Julia Buttd,NicoleBrennerd,AngelikaMicheld, Karin Lundbergb,g, Leonid Padyukovb,g, Ingrid E. Lundbergb,g, Elisabet Svenungssong, Ingemar Ernbergh, Sigurgeir Olafssoni, Alexander T. Diltheyj,k, Jan Hillerta, Lars Alfredssonl,m, Peter Sundströme, Peter Nilssonc,2, Tim Waterboerd,2, Tomas Olssona,b,2, and Ingrid Kockuma,b,2 aNeuroimmunology Unit, The Karolinska Neuroimmunology & Multiple Sclerosis Centre, Department of Clinical Neuroscience, Karolinska Institute, 171 76 Stockholm, Sweden; bCentrum for Molecular Medicine, Karolinska University Hospital, 171 76 Stockholm, Sweden; cDivision of Affinity Proteomics, Department of Protein Science, SciLifeLab, KTH - Royal Institute of Technology, 171 21, Solna, Sweden; dInfections and Cancer Epidemiology, Infection, Inflammation and Cancer Research Program, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany; eDepartment of Pharmacology and Clinical Neuroscience, Umeå University, 901 85 Umeå, Sweden; fDivision of Cellular and Clinical Proteomics, Department of Protein Science, SciLifeLab, KTH - Royal Institute of Technology, 171 21, Solna, Sweden; gDivision of Rheumatology, Department of Medicine Solna, Karolinska Institutet, 171 76 Stockholm, Sweden; hDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institute, 171 77 Stockholm, Sweden; ideCODE Genetics, Amgen, IS-101 Reykjavik, Iceland; jWellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom; kInstitute of Medical Microbiology and Hospital Hygiene, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany; lInstitute of Environmental Medicine, Karolinska Institute, 171 77 Stockholm, Sweden; and mCentre for Occupational and Environmental Medicine, Stockholm County Council, 171 77 Stockholm, Sweden Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved July 8, 2019 (received for review February 13, 2019) Multiple sclerosis (MS) is a chronic inflammatory, likely autoim- For the present study, it is of particular relevance that a com- mune disease of the central nervous system with a combination of bination of DRB1*15:01 carriage and high levels of Epstein-Barr genetic and environmental risk factors, among which Epstein-Barr virus nuclear antigen 1 (EBNA1) antibodies, primarily directed INFLAMMATION virus (EBV) infection is a strong suspect. We have previously toward 2 EBNA1 peptide fragments [aa 385 to 420 and aa 402 to IMMUNOLOGY AND identified increased autoantibody levels toward the chloride- 502], increase the risk of developing MS 10-fold (8, 9). Since more channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody than 95% of healthy individuals show an immune response to reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was EBV, it cannot be the sole cause of MS. However, it could be a measured using bead-based multiplex serology in plasma samples prerequisite for the disease and interact with other risk factors. from 8,746 MS cases and 7,228 controls. We detected increased anti- The mechanisms are far from clear. One hypothesis is molecular P = × −36 ANO2 antibody levels in MS ( 3.5 10 ) with 14.6% of cases mimicry (10). There are descriptions of T cell responses primarily and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 Significance known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; We have previously demonstrated an increased autoantibody 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 reactivity to Anoctamin 2 (ANO2), an ion channel expressed in and EBNA1 peptides demonstrated antibody cross-reactivity, map- the central nervous system (CNS), in multiple sclerosis (MS). We ping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene now show that ANO2 antibodies recognize a fragment of region was associated with anti-ANO2 antibody levels and HLA- Epstein-Barr virus (EBV) nuclear antigen 1, thereby constituting DRB1*04:01 haplotype was negatively associated with ANO2 sero- an example of molecular mimicry. In this way, the immune positivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels response toward EBV may take part in and promote CNS in- were not increased in patients from 3 other inflammatory disease flammation, likely through T cells reactive with the same pro- cohorts. The HLA influence and the fact that specific IgG production tein. In our very large case-control cohort, we demonstrate that usually needs T cell help provides indirect evidence for a T cell ANO2 the presence of ANO2 reactivity associates with a high MS risk, autoreactivity in MS. We propose a hypothesis where immune re- in particular together with HLA risk variants and high EBNA1 activity toward EBNA1 through molecular mimicry with ANO2 con- antibody titers, which we consider a strong argument for its tributes to the etiopathogenesis of MS. relevance in MS ethiopathogenesis. Anoctamin 2 | ANO2 | multiple sclerosis | molecular mimicry | Author contributions: P.N., T.W., T.O., and I.K. designed research; K.T., J. Huang, C.H., P.K., B.A., J.B., N.B., A.M., P.N., T.W., T.O., and I.K. performed research; M.B., I.L.B., K.L., L.P., Epstein-Barr virus I.E.L., E.S., I.E., S.O., A.T.D., J. Hillert, L.A., and P. Sundström contributed new reagents/ analytic tools; K.T., J. Huang, C.H., P.K., B.A., P. Stridh, J.B., N.B., A.M., P.N., T.W., T.O., and ultiple sclerosis (MS) is a chronic inflammatory disease of I.K. analyzed data; and K.T., J. Huang, C.H., P. Stridh, S.O., P.N., T.W., T.O., and I.K. wrote the paper. Mthe central nervous system (CNS) characterized by damage – Conflict of interest statement: Outside this work, T.O. has received unrestricted MS re- to myelin and neurons/axons (1 3) often with onset during young search grants, lecture and/or advisory board honoraria from: Biogen, Novartis, Merck, adulthood. Etiology involves both genetic and environmental risk Sanofi, and Roche. Outside this work, P.K. is working at Roche Diagnostics in factors and several of these have been shown to jointly and in- unrelated projects. teractively associate with increased risk for disease (4, 5). The This article is a PNAS Direct Submission. strongest genetic association is with the HLA gene region on Published under the PNAS license. chromosome 6p21, which harbors a series of class II risk alleles 1To whom correspondence may be addressed. Email: [email protected]. (e.g., DRB1*15:01), as well as class I alleles (e.g., A*02:01) that 2P.N., T.W., T.O., and I.K. contributed equally to this work. have been found to affect the risk of MS (2, 6, 7). Among various This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lifestyle/environmental factors thought to affect the risk of MS, 1073/pnas.1902623116/-/DCSupplemental. Epstein-Barr virus (EBV) infection is a strong candidate. Published online August 2, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1902623116 PNAS | August 20, 2019 | vol. 116 | no. 34 | 16955–16960 Downloaded by guest on September 28, 2021 against EBNA1 that cross-react with CNS/myelin components (11), but the mere existence of these does not inform us about their etiopathogenetic role. Well-known features of MS, such as ANO2DRB1*15:01A*02:01EBNA1 the association with HLA class II alleles (6), similarly demyelinating disease in the CNS of antigen-induced rodent models (12), reduced disease activity with immunomodulatory treatments (13), and even increased numbers of T cells producing proinflammatory cytokines in response to CNS antigens (14, 15) strongly support, but do not prove, a role of an autoimmune response to self-antigens in the CNS. Defining reliable MS-specific autoantigens has proven diffi- cult, which may partly be explained by epitope spreading (16) and the lack of validated assays for CNS antigen-specific T cells (17). It has been notoriously difficult to replicate findings of suggested autoantibodies in MS, despite the fact that demyelinating an- 011 510252035305 tibodies with unknown specificity are present (18). Nevertheless, Odds Ratio the identification of MS-specific antigenic targets is essential for understanding MS pathogenesis. Fig. 1. Odds ratios for MS with different combinations of risk factors. Risk We have previously identified increased autoantibody re- factors include ANO2 seropositivity, DRB1*15:01 carrier, A*02:01 noncarrier, and EBNA1-high (anti-EBNA1 antibody levels above median in controls). Red activity against Anoctamin 2 (ANO2) in an antibody screening of box indicates that the group of individuals is exposed to the risk factor and potential MS autoantigens with protein fragments representing white box that they are not. The ORs were calculated by comparison with a ∼38% of all human proteins (19). This finding was later replicated reference group, carrying none of the risk factors. Total numbers of MS cases where anti-ANO2 antibody levels were 5.3-fold higher in MS cases and controls in each group and P values are presented in SI Appendix, Table + than in controls (20). ANO2 is a Ca2 activated chloride channel S3. Bars in the graph indicate 95% confidence intervals. important in, e.g., transepithelial ion transport, smooth muscle contraction, olfaction, phototransduction, nociception, and con- −95 trol of neuronal excitability (21). We have previously shown that on anti-ANO2 antibody levels (P = 7.1 × 10 ), followed by −43 −28 neurons and glial cells from normal hippocampal and cortical study type (P = 1.1 × 10 ), MS status (P = 7.7 × 10 ), and age −21 regions express ANO2 and a clear increase in ANO2 staining (P = 4.6 × 10 ).